Analytical method for protein mapping using hydrogen/deuterium exchange

ABSTRACT

Analytical methods using hydrogen/deuterium exchange are provided which reduce or eliminate the back-exchange of deuterium for hydrogen. The methods, which are useful in protein and peptide mapping, include the steps of (a) providing a peptide or protein comprising a solvent accessible hydrogen; (b) exchanging the solvent accessible hydrogen for a deuterium; (c) separating the peptide or protein with supercritical fluid chromatography; and (d) analyzing by mass spectrometry the mass of the separated peptide or protein. Supercritical fluid chromatography enables the observation of fast exchanging hydrogen atoms missed using conventional liquid chromatography methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/771,542, filed Feb. 8, 2006. The application is incorporated hereinby reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. government support under Contract No.CHE-99-09502 awarded by the National Science Foundation. The U.S.government has certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention is generally in the field of protein or peptide mapping,and more particularly relates to mapping that utilizeshydrogen/deuterium exchange.

Proteins contain covalently bonded hydrogen atoms that are known toexchange with hydrogen atoms found in a surrounding solvent. Thus, ifthe solvent surrounding a protein is changed from normal water (H₂O) toheavy water (D₂O), the exchange of hydrogen (¹H) for deuterium (²H) canbe observed. This exchange reaction, known as hydrogen-deuteriumexchange (HDX), increases the molecular mass of the protein because thedeuterium nucleus is heavier than the common proton. Since thehydrogen-deuterium exchange rate reflects the exposure level of theparticular hydrogen atom to the solvent, spectrometric analysis can beutilized subsequent to HDX to determine which portions of the proteinhave increased in molecular mass and thus are accessible to thesurrounding solvent. This information can be extremely useful inunderstanding protein conformation, protein/protein interactions, andprotein/ligand interactions See Wang, et al., Biochemistry 37:15289-99(1998). For example, this “mapping” information may be used to determineprotein-ligand binding sites and is used in the pharmaceutical industryfor intelligent drug design.

Determining which portions of a protein have increased in molecular massdue to HDX (and thus which portions of a protein are in contact with thesurrounding solvent) generally requires a protein digestion step, adesalting/separation step, and a spectrometric analysis step. Currently,the separation technology used in HDX experiments is reversed phase highperformance liquid chromatography (RP-HPLC). Unfortunately, the mobilephase used in RP-HPLC is predominately water. The presence of water is amajor drawback because it allows for the back-exchange of deuterium forhydrogen during the separation step immediately prior to thespectrometric analysis. Back-exchange is a major problem with solutionHDX, as the back-exchange undesirably reduces both the amount and theresolution of the resulting data. It would therefore be useful toprovide a method that eliminates, or at least substantially reduces,back-exchange during separation.

In order to limit back-exchange, RP-HPLC conventionally is performed at0-4° C. and under very fast gradients in order to limit the period inwhich the sample is in contact with the aqueous phase. Unfortunately,this separation method reduces the reversed-phase chromatographicresolution, and increases the complexity of the spectra. Althoughreduced chromatographic resolution can be partially offset through theuse of high resolution Fourier transform ion cyclotron resonance massspectrometry (FT-ICR MS), these techniques still allow for the backexchange of deuterium for hydrogen. It therefore would be desirable toeliminate, or at least substantially reduce, back-exchange duringseparation while maintaining a high degree of chromatographicresolution.

BRIEF SUMMARY OF THE INVENTION

Analytical methods and apparatus which use hydrogen/deuterium exchangeare provided. In one aspect an analytical method is provided thatincludes the steps of: (a) providing a protein or peptide, which proteinor peptide comprises at least one solvent accessible hydrogen; (b)exchanging the at least one solvent accessible hydrogen for at least onedeuterium to make a deuterated protein or peptide; (c) usingsupercritical fluid chromatography to separate the deuterated protein orpeptide; and (d) analyzing the mass of the separated protein or peptide.The analysis of step (d) preferably includes mass spectrometry. Themethod may further include the step of digesting the peptide or proteinafter step (b) and before step (c).

The step of exchanging the at least one solvent accessible hydrogen forthe at least one deuterium may comprise exposing the peptide or proteinto a deuterated buffer. In one particular embodiment, the step of usingsupercritical fluid chromatography to separate the deuterated peptide orprotein is conducted at about 1° C.

The supercritical fluid chromatography preferably utilizes a mobilephase that comprises a supercritical fluid, such as carbon dioxide. Inanother embodiment, the supercritical fluid chromatography utilizes amobile phase that comprises a gas at a non-supercritical state ofenhanced fluidity. The supercritical fluid chromatography may utilize amobile phase modifier that comprises a polar or non-polar solvent.Examples of possible solvents include acetonitrile, dimethyl sulfoxide,dimethyl formamide, tetrahydroforan, dimethyl acetamide,trichloroethane, acetone, ethanol, methanol, isopropanol, ethyl acetate,formic acid, water, and mixtures thereof.

In one particular embodiment, an analytical method usinghydrogen/deuterium exchange is provided that includes the steps of: (a)obtaining a protein or peptide, which protein or peptide comprises atleast one solvent accessible hydrogen; (b) exposing the peptide orprotein to a deuterated buffer solution to effect exchanging the atleast one solvent accessible hydrogen for at least one deuterium,thereby making a deuterated protein or peptide; (c) mixing thedeuterated protein or peptide with a peptidase to produce a digesteddeuterated protein or peptide; (d) using supercritical fluidchromatography to separate the digested deuterated protein or peptide;and (e) using mass spectrometry to analyze the mass of the separatedprotein or peptide obtained from step (d).

In another aspect, an apparatus is provided for protein or peptideanalysis, which apparatus includes a supercritical fluid chromatograph;and a mass spectrometer, wherein the hydrogen-deuterium exchange iscoupled to the mass spectrometer so that the supercritical fluidchromatograph can separate and desalt a protein or peptide sample priorto mass analysis of the protein or peptide sample by the massspectrometer. The apparatus may further include robotic means forcarrying out hydrogen-deuterium exchange on the protein or peptidesample and for feeding the deuterated sample to the supercritical fluidchromatograph. The mass spectrometer may be equipped with anelectrospray ionization source. The apparatus may further include meansfor cooling the protein or peptide sample.

In another aspect, a method of protein or peptide mapping is providedthat includes the steps of: (a) selecting a non-deuterated protein orcomplex to be mapped, which protein or complex comprises at least onesolvent accessible hydrogen; (b) exchanging the at least one solventaccessible hydrogen for at least one deuterium to make a deuteratedprotein or peptide; (c) using supercritical fluid chromatography toseparate the deuterated protein or peptide; (d) analyzing the mass ofthe separated protein or peptide; and (e) comparing mass informationfrom the non-deuterated protein or complex with mass information fromthe separated protein or peptide to yield information about thestructure of the protein or complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a mass spectrum of a non-deuterated peptide followingsupercritical fluid chromatography at room temperature.

FIG. 1B is a mass spectrum of a deuterated peptide without anychromatography.

FIG. 1C is a mass spectrum of a deuterated peptide followingsupercritical fluid chromatography at room temperature.

FIG. 1D is a mass spectrum of a deuterated peptide followingsupercritical fluid chromatography at 1° C.

FIG. 2 is a graph comparing the level of deuterium uptake at varioustimes for a myoglobin peptic fragment having undergone SFC-TOF,HPLC-FT-ICR at 9.4 T, and HPLC-FT-ICR at 14.5 T.

FIG. 3 is an illustration and process flow diagram of one embodiment ofthe method and apparatus for carrying out the analytical processesdescribed herein.

DETAILED DESCRIPTION OF THE INVENTION

Improved methods and apparatus have been developed for protein orpeptide mapping by hydrogen/deuterium exchange. Studies have shown thatconventional HDX methods lose up to 90% of the data at the earlieststages of H/D exchange. It has now been discovered that by usingsupercritical fluid chromatography (SFC) as the peptideseparation/desalting technique following hydrogen/deuterium exchange,one advantageously can reduce the back exchange of deuterium forhydrogen to near immeasurable levels. See Emmett, et al. Anal. Chem.78:7058-60 (2006).

This reduction of back exchange can improve the spatial resolution ofthe protein-solvent interface by increasing the amount of data that isobtained by the spectrometric analysis. The mobile phase in SFC ispredominately a gas that is in a supercritical or enhanced fluiditystate. Preferably, the mobile phase has no exchangeable hydrogens andthus essentially eliminates the back-exchange observed in conventionalRP-HPLC separations. It also has been beneficially discovered that thesemethods and apparatus allow access to crucial structural informationthat has been lost in HDX experiments that use conventional separationmethods. By using supercritical fluid chromatography instead of highperformance liquid chromatography, the faster exchanging hydrogen atomsthat have been missed in previous HDX experiments are now observable.

The Methods

Protein or peptide mapping or other analysis is performed using HDX,while eliminating or reducing the back-exchange of deuterium forhydrogen. In a preferred embodiment, the analytical methods include thesteps of (a) providing a protein or peptide, which protein or peptidecomprises at least one solvent accessible hydrogen; (b) exchanging theat least one solvent accessible hydrogen for at least one deuterium tomake a deuterated protein or peptide; (c) using supercritical fluidchromatography to separate the deuterated protein or peptide; and (d)analyzing the mass of the separated protein or peptide.

The methods described herein are useful in mapping essentially anyprotein or peptide. As used herein, the term “peptide” generally refersto a linear chain of around 2 to 40 amino acids joined together withpeptide bonds, and the term “protein” generally refers a linear chain ofaround 30 or more amino acids joined together with peptide bonds. Theterm “solvent accessible hydrogen” refers to a hydrogen atom located ina position in the protein or peptide such that in solution, the hydrogenatom is exposed to the solvent surrounding the protein or peptide. Theamide hydrogen (hydrogen bonded to the amide nitrogen) is theexchangeable hydrogen that is most commonly monitored in HDXexperiments.

The step of exchanging the solvent accessible hydrogen for a deuteriumcan be done using essentially any process that effects the exchange ofaccessible hydrogen atoms for deuterium atoms. In one embodiment, thesolvent accessible hydrogen may be exchanged for a deuterium by exposinga solution of a peptide or protein by dilution of the protein sampleinto a deuterated buffer. Typically a 1/10 or 1/20 dilution is made,although dilution ratios may be varied. The “deuterated buffer” isessentially any solution in which the ratio of deuterium (²H) tohydrogen (¹H) is greater than the ratio of deuterium to hydrogen in theprotein or peptide. In a preferred embodiment, the protein/proteincomplex is digested by a peptidase to produce smaller peptide fragments.Suitable peptidases are known in the art.

The step of separating the deuterated peptides or proteins usingsupercritical chromatography generally involves a separation orpurification technique that includes feeding the deuterated peptides orproteins into/through the stationary phase using a supercritical fluidas the mobile phase, to yield separated proteins and peptides. As usedherein, the phrase “separating the peptide or protein” refers to thecomplete or partial isolation of least one peptide or protein from atleast one other salt, solvent, peptide, or protein, and the term“separated peptide or protein” refers to at least one peptide or proteinthat has been completely or partially isolated from at least one othersalt, solvent, peptide, or protein.

As used herein, the term “supercritical fluid” refers to essentially anysuitable mobile phase at a temperature and pressure above itsthermodynamic critical point. In another embodiment, the mobile phasemay consist of a gas at a non-supercritical state of enhanced fluidity.In various embodiments, the mobile phase may comprise carbon dioxide,nitrous oxide, or other gas that does not have an exchangeable hydrogen.The mobile phase may further include a mobile phase modifier, such as apolar or non-polar solvent. Non-exchangeable solvents are preferred.Examples of solvents that may be useful in the methods described hereininclude acetonitrile, dimethyl sulfoxide, dimethyl formamide,tetrahydroforan, dimethyl acetamide, trichloroethane, acetone, ethanol,methanol, isopropanol, ethyl acetate, formic acid, water, and mixturesthereof. In one embodiment, the deuterated protein or peptide undergoesa digestion step before the step of separating the peptides or proteinswith supercritical fluid chromatography. Analysis of smaller peptidefragments enhances the resolution of the location in the protein inwhich the deuterium has been incorporated, thus permitting a betterassessment of the solvent accessibility of the intact protein/proteincomplex.

The step of analyzing the mass of the separated peptide or protein maycomprise essentially any suitable technique known in the art. In apreferred embodiment, the step includes using mass spectrometry todetermine the masses of the separated deuterated proteins and peptides.Analytical techniques and equipment for this are well known in the art.High resolution mass spectrometry instrumentation (FT-ICR MS) ispreferred, but other lower resolution instruments such as quadrupole,TOF, ion traps or orbitraps may also be used in the analysis. The methodof sample ionization may also vary, in this embodiment electrosprayionization was used.

The information obtained from analyzing the mass of the peptide orprotein can be extremely useful in understanding protein confirmation,protein/protein interactions, and protein/ligand interactions. Forexample, HDX coupled to SFC can be used to characterize proteininteractions with other molecules. By performing hydrogen-deuteriumexchange on both the isolated protein and the protein bound to anothermolecule, the region of the protein that is protected from the solventby the other molecule can be observed. This data can be used to build amodel of how the protein interacts with the molecule, which can beextremely valuable for intelligent drug design. By using supercriticalfluid chromatography to eliminate or reduce the back-exchange ofdeuterium for hydrogen, the fastest exchanging hydrogen atoms that havebeen missed in previous HDX experiments can be observed to provide abetter understanding of which protein regions interact with the othermolecule. Furthermore, HDX coupled to SFC can be used to characterizethe folding pathway of proteins. By refolding a protein while performinghydrogen-deuterium exchange, the parts of the protein that fold firstwill be protected from the deuterated solvent more quickly than theareas of the protein that fold later. By using supercritical fluidchromatography to eliminate or reduce the back-exchange of deuterium forhydrogen, the resolution of this information can be increased to providea better understanding about how the protein folds.

The Apparatus

One skilled in the art can readily adapt and connect conventionalchromatography equipment and materials together with a massspectrometer, suitably configured to practice the methods describedherein. The ionization source should be configured to eliminate thefreezing of the SFC eluent as it changes from a supercriticalfluid/enhanced fluid to a gas. In one embodiment, the SFC eluent ispost-column split to reduce the flow rate to the ion source of the massspectrometer and the ion source is heated to eliminate freezing of theSFC mobile phase. The apparatus comprises a supercritical fluidchromatograph operably coupled to a mass spectrometer. One embodiment isillustrated in FIG. 3.

The supercritical fluid chromatography system includes a back pressureregulating device to maintain the system pressure above the criticalpressure (Pc) of the fluid used, from a delivery pump through to thedetector. The back pressure device may be a simple restrictor (e.g., anorifice) or a mechanical or electronic feedback regulator. The systemmay include a thermostat and heat exchanger means (e.g., a chiller) toaid in start-up and/or in helping to keep the temperature of the mobilephase fluid above its critical temperature (Tc).

When the mobile phase includes a solvent with an exchangeable hydrogen,then the apparatus preferably includes a cooling component for keepingthe deuterated protein or peptide sample chilled throughout the steps ofsample quenching, dilution, digestion, SFC injection, separation, and tothe mass spectrometry analysis, so that back exchange is minimized.

In one embodiment, the apparatus includes Fourier-transform ioncyclotron resonance mass spectrometry (FT-ICR). The apparatus can bemade completely automated. In one embodiment, the apparatus can be aspecific solution HDX system utilizing robotics coupled to SFC with massspectrometric analysis.

The methods and apparatus described above will be further understoodwith reference to the following non-limiting examples.

EXAMPLES

The following materials and equipment were used in the examples: HDX andsample injections were performed with a LEAP robot (Carboro, N.C.)programmed with interlaced software developed at the National HighMagnetic Field Laboratory (NHMFL). SFC was performed with a Jasco system(Easton, Md.) consisting of a PU-1580 CO₂ delivery pump, a PU-1580Intelligent HPLC pump (for modifier delivery) and a BD-158-81 backpressure regulators. SFC effluent was monitored by a Waters Micromass(Milford, Mass.) LCT ESI-TOF mass spectrometer equipped with a Z-sprayESI source. Each HDX sample (10 μL at 40 M) was separated on a 4.6 mm×50mm Waters Atlantis HILIC 5 micron silica column. Gradient elution wasperformed from 80% CO₂ to 40% CO₂ in 4 minutes at 3.5 mL/min. The mobilephase modifier was 40% acetonitrile, 40% methanol, 19% H₂O, and 1%formic acid.

Example 1 HDX-SFC of a Small Peptide

The smallest peptic fragment normally produced by the digestion of aprotein is 5 amino acids in length. Thus, a synthetic penta-peptideIFVQK was utilized to test for the retention of a small peptic fragmenton a SFC column. IFVQK has 4 exchangeable amide hydrogens, and anon-deuterated mass-to-charge ratio (m/z) of 634.3.

SFC-MS was first performed on the non-deuterated peptide at roomtemperature. As seen in FIG. 1A, running the non-deuterated peptidethrough the SFC column coupled to the mass spectrometer produced aspectrum with a peak appearing at 634.3 m/z, demonstrating that a smallpeptide can be retained on a SFC column.

Next, the fully deuterated peptide was electrosprayed directly from adeuterated solvent (i.e., with side chains also fully deuterated) intothe mass spectrometer. As seen in FIG. 1B, the spectrum featured anaverage mass-to-charge ratio that was higher than 634.3 m/z due to thedeuterium increasing the molecular weight of the peptide.

SFC-MS was then performed on the fully deuterated peptide at roomtemperature (22° C.). As seen in FIG. 1C, the spectrum of the elutedpeptide had an isotopic distribution number-average m/z of about 636,indicating an average incorporation of two deuteriums.

Finally, SFC-MS was performed on the fully deuterated peptide at 1° C.and with a modified gradient (modifier held at 1° C. and supplementedwith 1% formic acid). As seen in FIG. 1D, running the non-deuteratedpeptide through the SFC column under these conditions produced aspectrum with an isotopic distribution number-average m/z of about 638,indicating an average incorporation of the maximum possible fourdeuteriums from the exchangeable amide hydrogens. This result indicatesnear zero back-exchange of deuterium for hydrogen.

Example 2 HIDX-SFC of Myoglobin

Deuterated myoglobin protein was digested into peptic fragments.(Myoglobin Peptic Fragment (aa. 56 to 69) KASELDLKKHGTVVL,m/z=[762.9376]²⁺) Deuterium uptake was then calculated using threedifferent techniques: (1) Supercritical fluid chromatography followed bytime of flight mass spectrometry (SFC-TOF); (2) HPLC-FT-ICR at 9.4 T;and (3) HPLC-FT-ICR at 14.5 T. As seen in FIG. 2 and Table 1, deuteriumincorporation was much higher in the SFC separated peptides than in theHPLC separated peptides at all time points in the experiment due to thereduced back exchange of deuterium for hydrogen. Furthermore, the SFCseparated peptides had the most dramatic increase in deuteriumincorporation during the earliest time points of the H/D exchange, whichdemonstrates how this method allows access to crucial information thathas been lost in HDX experiments that use conventional separationmethods. By using supercritical fluid chromatography instead of highperformance liquid chromatography, the faster exchanging hydrogen atomsthat appear during earlier time points are now observable.

TABLE 1 SFC-TOF HPLC 9.4 T HPLC 14.5 T Av. D. Av. D. Av. D. Mass SDuptake Mass SD uptake Mass SD Uptake 0Ctrl 1526.316 0.02 0.0 1526.0120.12 0.0 1526.636 0.02 0.0 30 1528.632 0.66 2.3 1526.553 0.08 0.51527.437 0.22 0.8 60 1529.363 0.36 3.0 1526.403 0.08 0.4 1527.161 0.310.5 120 1529.363 1.57 3.0 1526.348 0.14 0.3 1527.638 0.24 1.0 2401530.703 1.44 4.4 1526.420 0.08 0.4 1527.449 0.01 0.8 480 1530.562 0.414.2 1526.470 0.14 0.5 1527.866 0.05 1,2 900 1529.974 0.19 3.7 1526.9140.22 0.9 1528.170 0.27 1.5 1800 1530.547 0.15 4.2 1527.227 0.18 1.21528.413 0.29 1.8 3600 1530.873 0.08 4.6 1527.528 0.14 1.5 1528.484 0.231.8

Publications cited herein and the materials for which they are cited arespecifically incorporated by reference. Modifications and variations ofthe methods and devices described herein will be obvious to thoseskilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe appended claims.

1. An analytical method using hydrogen/deuterium exchange, comprisingthe steps of: (a) providing a protein or peptide, which protein orpeptide comprises at least one solvent accessible hydrogen; (b)exchanging the at least one solvent accessible hydrogen for at least onedeuterium to make a deuterated protein or peptide; (c) usingsupercritical fluid chromatography to separate the deuterated protein orpeptide; and (d) analyzing the mass of the separated protein or peptide.2. The method of claim 1, further comprising the step of digesting thepeptide or protein after step (b) and before step (c).
 3. The method ofclaim 1, wherein the step of exchanging the at least one solventaccessible hydrogen for the at least one deuterium comprises exposingthe peptide or protein to a deuterated buffer.
 4. The method of claim 1,wherein the step of using supercritical fluid chromatography to separatethe deuterated peptide or protein is conducted at about 1° C.
 5. Themethod of claim 1, wherein the supercritical fluid chromatographyutilizes a mobile phase that comprises a supercritical fluid.
 6. Themethod of claim 5, wherein the mobile phase comprises carbon dioxide. 7.The method of claim 1, wherein the supercritical fluid chromatographyutilizes a mobile phase modifier that comprises a polar or non-polarsolvent.
 8. The method of claim 7, wherein the solvent is selected fromthe group consisting of comprises acetonitrile, dimethyl sulfoxide,dimethyl formamide, tetrahydroforan, dimethyl acetamide,trichloroethane, acetone, ethanol, methanol, isopropanol, ethyl acetate,formic acid, water, and mixtures thereof.
 9. The method of claim 1, thesupercritical fluid chromatography utilizes a mobile phase modifier thatcomprises a polar or non-polar solvent.
 10. The method of claim 1,wherein the step of analyzing the mass of the separated peptide orprotein comprises mass spectrometry.
 11. An analytical method usinghydrogen/deuterium exchange, comprising the steps of: (a) obtaining aprotein or peptide, which protein or peptide comprises at least onesolvent accessible hydrogen; (b) exposing the peptide or protein to adeuterated buffer solution to effect exchanging the at least one solventaccessible hydrogen for at least one deuterium, thereby making adeuterated protein or peptide; (c) mixing the deuterated protein orpeptide with a peptidase to produce a digested deuterated protein orpeptide; (d) using supercritical fluid chromatography to separate thedigested deuterated protein or peptide; and (e) using mass spectrometryto analyze the mass of the separated protein or peptide obtained fromstep (d).
 12. The method of claim 11, wherein the supercritical fluidchromatography utilizes (i) a mobile phase that comprises asupercritical fluid which consists essentially of carbon dioxide, and(ii) a mobile phase modifier which comprises a polar or non-polarsolvent.
 13. A method of protein or peptide mapping comprising using theresults of the analytical method of claim 1 to determine structuralproperties of a peptide or protein.
 14. A method of protein or peptidemapping comprising using the results of the analytical method of claim11 to determine structural properties of a peptide or protein.
 15. Amethod of protein or peptide mapping comprising: (a) selecting anon-deuterated protein or complex to be mapped, which protein or complexcomprises at least one solvent accessible hydrogen; (b) exchanging theat least one solvent accessible hydrogen for at least one deuterium tomake a deuterated protein or peptide; (c) using supercritical fluidchromatography to separate the deuterated protein or peptide; (d)analyzing the mass of the separated protein or peptide; and (e)comparing mass information from the non-deuterated protein or complexwith mass information from the separated protein or peptide to yieldinformation about the structure of the protein or complex.